Stokes Shift Spectroscopy (S3) offers a novel way to rapidly measure spectral fingerprints of complex molecular mixtures in tissue. The changes of key fluorophores from normal state to the malignant state can be reflected by alteration of Stokes Shift Spectra (S3 spectra). S3 measurements can be used to acquire enough information of different key fluorophores from one spectrum to speed up spectral acquisition time. In this study, we demonstrate the usefulness of the S3 technique to distinguish the malignant tissue from the normal prostate and breast tissues. The optimal wavelength shift constant (Δλc) of S3 spectra measurements for prostate/breast cancer detection were determined to be 40 nm. The underlying physical and biological basis for S3 is discussed. For the first time, our work explicitly discloses how and why S3 is supreme in comparison with other conventional spectroscopic techniques.
Stokes shift spectroscopy (S3) offers a novel and simpler way to rapidly recognize spectral fingerprints of multiple fluorophores in complex media such as in tissue. This spectroscopic technique can be used as an effective approach to detect cancer in tissue. The alterations of the measured S3 spectra between cancerous and normal tissues were observed in human breast and prostate samples. In order to obtain the optimal Stokes shift interval, Δλ i , for the purpose of breast/prostate cancer detection using S3, the S3 spectra of a mixed aqueous solution of tryptophan, nicotinamide adenine dinucleotide, and flavin were measured with different Δλ i values. The experimental results analyzed using nonnegative least square method show that there is a reduced contribution from collagen and an increased contribution from tryptophan to the S3 signal of the cancerous tissue as compared with those of the normal tissue. This study indicates that the changes of relative contents of tryptophan and collagen in tissue shown by the S3 spectra may present potential native biomarkers for breast and prostate cancer detection. S3 has the potential to be a new armamentarium.
KEYWORDS: Cancer, Tissues, Collagen, Principal component analysis, Luminescence, Signal detection, Breast cancer, Breast, Fluorescence spectroscopy, Biological research
A novel approach to cancer detection in biomarkers spectral subspace (BSS) is proposed. The basis spectra of the subspace spanned by fluorescence spectra of biomarkers are obtained by the Gram-Schmidt method. A support vector machine classifier (SVM) is trained in the subspace. The spectrum of a sample tissue is projected onto and is classified in the subspace. In addition to sensitivity and specificity, the metrics of positive predictivity, Score1, maximum Score1, and accuracy (AC) are employed for performance evaluation. The proposed BSS using SVM is applied to breast cancer detection using four biomarkers: collagen, NADH, flavin, and elastin, with 340-nm excitation. It is found that the BSS SVM outperforms the approach based on multivariate curve resolution (MCR) using SVM and achieves the best performance of principal component analysis (PCA) using SVM among all combinations of PCs. The descent order of efficacy of the four biomarkers in the breast cancer detection of this experiment is collagen, NADH, elastin, and flavin. The advantage of BSS is twofold. First, all diagnostically useful information of biomarkers for cancer detection is retained while dimensionality of data is significantly reduced to obviate the curse of dimensionality. Second, the efficacy of biomarkers in cancer detection can be determined.
Cancer cells display high rates of glycolysis even under normoxia and mostly under hypoxia. Warburg proposed this
effect of altered metabolism in cells more than 80 years ago. It is considered as a hallmark of cancer. Optical
spectroscopy can be used to explore this effect.
Pathophysiological studies indicate that mitochondria of cancer cells are enlarged and increased in number. Warburg
observed that cancer cells tend to convert most glucose to lactate regardless of the presence of oxygen. Previous
observations show increased lactate in breast cancer lines.
The focus of this study is to investigate the relative content changes of lactate and mitochondria in human cancerous and
normal breast tissue samples using optical spectroscopic techniques. The optical spectra were obtained from 30
cancerous and 25 normal breast tissue samples and five model components (Tryptophan, fat, collagen, lactate and
mitochondrion) using fluorescence, Stokes shift and Raman spectroscopy. The basic biochemical component analysis
model (BBCA) and a set of algorithm were used to analyze the spectra.
Our analyses of fluorescence spectra showed a 14 percent increase in lactate content and 2.5 times increase in
mitochondria number in cancerous breast tissue as compared with normal tissue. Our findings indicate that optical
spectroscopic techniques may be used to understand Warburg effect. Lactate and mitochondrion content changes in
tumors examined using optical spectroscopy may be used as a prognostic molecular marker in clinic applications.
The microinjection of organelles, plants, particles or chemical solutions into Amoeba proteus coupled with spectroscopic
analysis and observed for a period of time provides a unique new model for cancer treatment and studies. The amoeba is
a eukaryote having many similar features of mammalian cells. The amoeba biochemical functions monitored
spectroscopically can provide time sequence in vivo information about many metabolic transitions and metabolic
exchanges between cellar organelles and substances microinjected into the amoeba. It is possible to microinject algae,
plant mitochondria, drugs or carcinogenic solutions followed by recording the native fluorescence spectra of these
composites. This model can be used to spectroscopically monitor the pre-metabolic transitions in developing diseased
cells such as a cancer. Knowing specific metabolic transitions could offer solutions to inhibit cancer or reverse it as well
as many other diseases.
In the present study a simple experiment was designed to test the feasibility of this unique new model by injecting algae
and chloroplasts into amoeba. The nonradiative dynamics found from these composites are evidence in terms of the
emission ratios between the intensities at 337nm and 419nm; and 684nm bands. There were reductions in the metabolic
and photosynthetic processes in amoebae that were microinjected with chloroplasts and zoochlorellae as well of those
amoebae that ingested the algae and chloroplasts. The changes in the intensity of the emissions of the peaks indicate that
the zoochlorellae lived in the amoebae for ten days. Spectral changes in intensity under the UV and 633nm wavelength
excitation are from the energy transfer of DNA and RNA, protein-bound chromophores and chlorophylls present in
zoochlorellae undergoing photosynthesis. The fluorescence spectroscopic probes established the biochemical interplay
between the cell organelles and the algae present in the cell cytoplasm. This hybrid state is indicative that a symbiotic
system is in place and the results definitely support the potential use of this unique new model. This model many help in
plant / animal and cancer processes.
We use the characteristic function method to obtain the probability distribution for the random force in the independent harmonic oscillator model. This model encompasses many important physical problems. The general characteristic function method is reviewed for the classical case and formulated for the quantum mechanical case. We obtain the probability distribution at one time and also the joint distribution of force for two different times.
The Stokes-shifted emission spectra were measured for various photoactive biomolecules such as tryptophan, collagen, NADH and flavin in aqueous solution and tissue. Information is obtained on the molecular activity in the tissue. This new approach allows for the extraction of information not obtained from excitation and/or fluorescence spectroscopy for a single spectral scan.
Breast tissues were investigated using diffuse reflectance spectroscopy to yield the absorption spectrum from Kubelka-Munk Function (KMF). A specified spectral feature measured in adipose tissue was assign to (beta) -carotene, which can be used to separate fat from other molecular components in breast tissues. The peaks of (KMF) at 260nm and 280nm were attributed to DNA and proteins.
Fluorescence images were acquired form gynecological tissues for multiple combinations of emission and excitation wavelengths in the UV and blue spectral regions. The wavelength combinations were selected to highlight different tissue molecules whose fluorescence signatures have been shown to potential in the detection of malignancy. These images were analyzed to determine the size, shape and location of different tissue structures.
Normal, fibroadenoma, malignant, and adipose breast tissues were investigated using Kubelka-Munk Spectral Function (KMSF). The spectral features in KMSF were identified and compared with absorption spectra determined by transmission measurements. A specified spectral feature measured in adipose tissue was assigned to (beta) -carotene, which can be used to separate fat form other molecular components in breast tissues. The peaks of (KMF) at 260nm and 280nm were attributed to DNA and proteins. The signal amplitude over 255nm to 265nm and 275nm to 285nm were found to be different for malignant fibroadenoma, and normal tissues.
Salient differences observed in the excitation spectra have been shown to reveal changes of proteins in the cellular and extracellular matrices of malignant tissues and degree of invasiveness
Fluorescence, excitation, diffuse reflectance and FT-IR Raman spectral scans of squamous cell carcinoma of the head and neck have been studied. Eleven specimens of squamous cell carcinoma in larynx, lymph node, throat, grand, soft palate and epiglotts and nine corresponding normal specimens was tested. The excitation spectral differences between squamous cell carcinoma and normal were investigated in the protein spectral range 260 nm to 300 nm. The ratios intensity at various excitation wavelengths were used as criterion to separate carcinoma and normal tissues. The t- test of these parameter have been made. The P values of all these three parameters are less than 0.005. The statistical results of sensitivity and specificity of using three parameters compared to pathological results are about 90 percent. The Raman spectroscopy was measured. Characteristics vibrational modes were used to separate out carcinoma and correlated with excitation spectroscopy data.
A novel method for correcting the fluorescence emission and excitation spectra is applied to native fluorescence spectra from normal and cancerous human breast tissues. The method effectively eliminates the distortions produced by internal light-absorption and allows a direct, real-time, correction without any iterative procedures. A simplified photon- diffusion model was used to develop the method. An analysis of both the true fluorescence spectra, and the diffuse reflectance spectra transformed into the ratio of absorption and reduced scattering coefficients, shows distinctive biochemical differences between cancerous and normal breast tissues. The fluorescence spectra feature a lower contribution of NADH and, possibly, collagen and elastin in cancerous tumor tissues as compared with normal tissues. The fluorescence spectra from cancerous tumors also show a lower degree of variability than the spectra from normal tissues. The corrected spectra from cancerous tumors show a greater similarity in their profiles than the non-corrected fluorescence spectra distorted by the internal light- absorption.
Fluorescence spectra of dermal lesions on 48 rats were investigated using excitation wavelengths of 275, 300, and 340 nm. Emissions at 340 and 460 nm were measured in both the forequarter and hindquarter lesions as function of time after incision. Unlike the 460-nm emission, intensity at 340 nm increased with time and then saturated. Control studies on intact skin and lesions in dead rats failed to demonstrate any time-dependent changes at these wavelengths. It appears that the increase in the 340-nm intensity is due to changes in the tryptophan level, and may reflect accumulation of fibrinogen in the early wound healing process.
We report on an analysis of diffuse reflectance spectra measured in conjunction with the fluorescence from normal human breast tissues and malignant breast tumors. The diffuse reflectance spectra from excised, air-equilibrated, human breast tissue samples show lower fractions of oxygenated hemoglobin and higher content of ferric (Fe3+) heme in malignant breast tumor samples than in normal breast tissues. Normal tissues are found to be easily deoxygenated and reoxygenated, but malignant tumors usually do not change their state as much. An analysis of tissue oxygenation parameters is discussed with respect to an enhancement of predictive power of fluorescence diagnostic method. The oxygenation state of tissues may be used as an additional marker in cancer diagnostics.
Native fluorescence spectroscopy of biomolecules has emerged as a new modality to the medical community in characterizing the various physiological conditions of tissues. In the past several years, many groups have been working to introduce the spectroscopic methods to diagnose cancer. Researchers have successfully used native fluorescence to distinguish cancerous from normal tissue samples in rat and human tissue. We have developed three generations of instruments, called the CD-scan, CD-ratiometer and CD-map, to allow the medical community to use optics for diagnosing tissue. Using ultraviolet excitation and emission spectral measurements on both normal and cancerous tissue of the breast, gynecology, colon, and aerodigestive tract can be separated. For example, from emission intensities at 340 nm to 440 nm (300 nm excitation), a statistically consistent difference between malignant tissue and normal or benign tissue is observed. In order to utilize optical biopsy techniques in a clinical setting, the CD-scan instrument was developed, which allows for rapid and reliable in-vitro and in-vivo florescence measurements of the aerodigestive tract with high accuracy. The instrumentation employs high sensitivity detection techniques which allows for lamp excitation, small diameter optical fiber probes; the higher spatial resolution afforded by the small diameter probes can increase the ability to detect smaller tumors. The fiber optic probes allow for usage in the aerodigestive tract, cervix and colon. Needle based fiber probes have been developed for in-vivo detection of breast cancer.
Fluorescence spectroscopy was applied to characterize normal, malignant and adipose breast tissues. Excitation, emission, and synchronized diffusive reflectance spectral scans were measured on over one hundred specimens for the purpose of developing an improved spectroscopic diagnostic technique. These techniques were able to successfully distinguish malignant tissue from adipose glandular fibrous and normal tissue. A sensitivity of 91% for fifty-six (56) malignant specimens with specificity of 91% for forty-six (46) benign tissue specimens has been achieved, using pathology as the golden standard.
Fluorescence spectra of tryptophan and bacteria were measured at different concentrations using a Mediscience CD-Scan unit. The emission spectra of tryptophan were obtained using an excitation wavelength at 280 nm. The excitation spectra were obtained at the emission of 340 nm. The minimum detectable concentration of tryptophan was found to be 10-8 M. The emission spectra for bacteria were probed at 340 nm. The minimum detectable number of bacteria in a beam of the excitation light was determined to be about 30. Assuming that the emission band at 340 nm of bacteria comes from tryptophan, the number of tryptophan per bacterium was estimated to be 108. This approach to determine the number is almost consistent with that obtained using a weight method.
Fluorescence spectra of dermal lesions on 48 rats have been investigated using excitation wavelengths of 275 nm, 300 nm and 340 nm. Emission at 340 nm and 460 nm were measured in both forequarter and hindquarter lesions. Unlike 460 nm emission, intensity at 340 nm increased with time and then saturated. Control studies on intact skin and lesions in dead rats failed to demonstrate any time dependent changes. It appears that the 340 nm intensity changes is due to changes in the tryptophan level, and may reflect a factor in the wound healing process.
The distribution, contents, and cellular uptake processes of various photosensitizers have been measured by scanning microspectrophotometry for single living cells of squamous epithelium carcinoma.
We used a dielectric microsphere model which is based on geometrical optics to describe the generation of trapping force by a single laser beam. Our results show that the microsphere can be trapped in some cases. For a typical microsphere, which has a 10 microns diameter, the trapping point is about 2 microns below the focusing point of the laser, and the trapping range is about 4 microns. The transverse trapping range is about 16 microns. And we find, that if we use suitable TEM mode of the laser beam, we can raise the trapping force for several times. This model can also be extended to biological cells.
In the present paper we address the question if fluorescence diagnostics can be used to monitor and possibly predict the outcome of photodynamic therapy (PDT) using the tumor seeking agents Photofrin and (delta) -aminolevulinic acid (ALA). The degree of selective uptake may vary from patient to patient and it would be interesting to use the drug-related fluorescence signal as a tool to tailor the treatment strategy. Clearly, the fluorescence intensity cannot be directly related to the tissue drug contents because of varying absorption and scattering properties of the tissue. However, because of the real-time capability of fluorescence it is interesting to see how far the fluorescence information can be utilized for optimizing the light delivery. Patients with basal cell carcinoma and spread metastatic breast cancer in the skin were treated. Two different doses, 1 and 2 mg/kg b.w. of Photofrin (Quadra Logic, Vancouver, Canada) were used. The treatment laser was a Nd:YAG pumped dye laser (Multilase Dye 600, Technomed International, Paris/Bron, France). The system provides 1064 nm IR and 532 nm green light from the Nd:YAG laser as well as red light in the region 620 - 670 nm from a dye laser. The treatment procedure was preceded by fluorescence measurements for allowing comparisons between the diagnostic signals and the treatment outcome. At the end of the treatment, fluorescence was again monitored to assess the degree of bleaching manifested by the appearance of an additional red peak. Our data on the connection between fluorescence signals, delivered dose and observed treatment outcome are presented and the potential of imaging fluorescence monitoring in PDT dosimetry is discussed.
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